1. (Field of the Invention)
The present invention generally relates to the art of centrifugal counter-current distribution chromatography and, more particularly, to an apparatus for carrying out the centrifugal counter-current distribution chromatography for the assay and the separation and purification, i.e. for the quantitative or qualitative analysis, of biochemical substances or natural organic compounds, and also for the refinement of crude samples.
2. (Description of the Prior Art)
When centrifugal counter-current distribution chromatography is continuously performed, if the stationary phase liquid and the mobile phase liquid are mixed and separated while centrifugally accelerated, the time required to complete the separation and the assay can be remarkably reduced and the limitation imposed by the distribution liquid system can be alleviated. Numerous apparatuses for performing centrifugal counter-current distribution based on this idea have been proposed, all of which make use of a coiled separation tube disposed in a centrifuge for facilitating the separation. The use of a coiled separation tube present a problem in that, if the quantity of the sample to be separated and analyzed is increased, the coiled separation tube must have a correspondingly increased inner diameter. As the inner diameter of the coiled separation tube increases the distribution of substances between, the mobile phase liquid and the stationary phase liquid correspondingly decrease, accompanied by an increased spread of the separation peak resulting from the diffusion. In view of this, the amount of samples that can be separated and processed with the coiled separation tube has been limited accordingly.
The assignee of the present invention has disclosed a centrifugal counter-current distribution chromatographic apparatus, which has successfully alleviated the above-discussed problems, in Japanese Patent Publication No. 58-1386, published in 1983. According to this publication, there is disclosed a cassette comprising a single separation passage and adapted to be detachably mounted on an outer peripheral portion of a motor-driven rotor, such as illustrated in FIGS. 17 to 20 of the accompanying drawings.
The prior art cassette disclosed in the above-mentioned publication and generally identified by 1 in FIGS. 17 to 20 comprises a generally elongated and generally rectangular cross-sectioned body 2 made of synthetic resin and having first and second opposite side faces to which respective metal side plates 3 are secured through sealing plates 4 by means of fastening members such as, for example, set screws. The elongated body 2 has defined therein a plurality of, for example two rows of, large-diameter bores 5a and 5b and an equal number of rows of small-diameter bores 6a and 6b. Each of the large-diameter bores 5a and 5b of each row extends substantially perpendicular to the longitudinal -axis of the elongated body 2 with its opposite ends open to the first and second side faces of the elongated body 2 and, similarly, each of the small-diameter bores 6a and 6b of each row extends substantially perpendicular to the longitudinal axis of the elongated body 2 with its opposite ends open to the first and second side faces of the elongated body 2. The large-diameter bores 5a and 5b and the small-diameter bores 6a and 6b communicate at their opposite ends, with each other through respective transverse grooves 7 defined in the first and second side faces of the elongated body 2 whereby, when the sealing plates 4 are respectively secured to the first and second side faces of the elongated body 2, a generally tortuous single separation passage extending from an inlet port 8 to an outlet port 9, which are both open at an upper end region of the first side face of the elongated body 2, is formed in the cassette 1. The tortuous passage so defined in the cassette 1 has a series of small-diameter passage portions, defined by the small-diameter bores 6a and 6b and the transverse grooves 7, and a series of large-diameter passage portions defined by the large-diameter bores 5a and 5b, said series of small-diameter and large-diameter passage portions alternately disposed over the entire length of the tortuous separation passage.
When in use, the cassette 1 having the construction described above is mounted on the motor-driven rotor with the small-diameter and large-diameter passage portions, that is, the small-diameter and large-diameter bores 6a, 6b and 5a, 5b, oriented in a radial direction radially of the motor-driven rotor and parallel to the direction in which a centrifugal force acts during high speed rotation of the motor-driven rotor. The inlet port 8 and the outlet port 9 respectively communicate with a source of a liquid medium to be examined and a collecting vessel through associated collets 10 and 11 to which suitable lengths of tubing are connected.
According to the prior art cassette 1, not only can a relatively large number of theoretically effective stages be employed, but also the volume of the stationary phase liquid retained in each of the large-diameter passage portions, that is, each of the large-diameter bores 5a and 5b, can be advantageously increased with a resulting increase of the ratio of volume between the stationary phase liquid and the mobile phase liquid. Because of these advantages, the use of the cassette 1 having the construction shown in and described with reference to FIGS. 17 to 20 is effective to accomplish the separation of samples in a reduced amount of time.
However, it has been found that the prior art cassette for use in centrifugal counter-current distribution chromatography is very complicated and, therefore, costly to fabricate. More specifically, not only is a time-consuming and complicated drilling operation required to form the enormous number of large-diameter and small-diameter bores in the elongated body, but also these large-diameter and small-diameter bores must be accurately positioned relative to each other, requiring a highly precise machining technique. Therefore, the prior art cassette is costly to fabricate and is, therefore, high-priced.